“Study on Blood Ethanol Stability in the Context of Driving under influence of Alcohol Investigations”

Driving under the influence of alcohol is a major public health and legal issue, as a substantial proportion of traffic accidents are associated with alcohol consumption. In Italy, Articles 186 and 186-bis of the Highway Code establish legal thresholds for blood alcohol concentration (BAC) and impose sanctions according to specific limits (0 g/L, 0.5 g/L, 0.8 g/L, and 1.5 g/L) based on driver categories. Following ethanol determination in such cases, blood samples are typically stored at −20 °C for at least one year to allow potential counter-analysis requested by judicial authorities or the individuals involved. However, because ethanol is volatile and susceptible to microbial activity after collection, its concentration may change over time, potentially affecting forensic interpretations—especially when initial results are near legal thresholds. This study evaluated the long-term stability of ethanol in blood samples collected under Articles 186 and 186-bis, stored at −20 °C for up to 30 months, and re-analyzed to verify analyte preservation and analytical reliability. A total of 780 blood samples collected between 2022 and 2023, each initially testing positive for ethanol (BAC > 0.05 g/L), were re-analyzed in 2024 using a validated Headspace Gas Chromatography–Flame Ionization Detection (HS-GC-FID) method. The limits of detection and quantification were 0.01 g/L and 0.05 g/L, respectively, and a ±10% analytical tolerance was applied. Results showed that approximately 60% of samples remained within this tolerance range, while 24% exhibited moderate decreases and 16% showed increases, mostly within the 10–20% range. A two-factor ANOVA indicated a significant effect of storage duration (p<0.05) but no significant influence of vacutainer type (p>0.05), demonstrating comparable performance between sodium fluoride and EDTA-based tubes. Overall, these findings confirm that ethanol concentrations remain stable for up to 30 months when samples are properly sealed and stored at −20°C, ensuring analytical reliability and legal defensibility in forensic counter-analyses.